1. Field of the Invention
The present invention relates to a method for reducing the cellular content and activity of NF-xcexaB by use of inhibitors of proteasome function or ubiquitin conjugation.
2. Description of Related Art
The transcription factor NF-xcexaB and other members of the rel family of protein complexes play a central role in the regulation of a remarkably diverse set of genes involved in the immune and inflammatory responses (Grilli et al., International Review of Cytology 143:1-62 (1993)). For example, NF-xcexaB is required for the expression of a number of immune response genes, including the Ig-xcexa light chain immunoglobulin gene, the IL-2 receptor xcex1 chain gene, the T cell receptor xcex2 chain gene, and class I and II major histocompatibility genes. In addition, NF-xcexaB has been shown to be required for a number of genes involved in the inflammatory response, such as the TNF-xcex1 gene and the cell adhesion genes, E-selectin, I-cam, and V-cam. NF-xcexaB is also required for the expression of a large number of cytokine genes such as IL-2, IL-6, G-CSF, and IFN-xcex2. Finally, NF-xcexaB is essential for the expression of the human immunodeficiency virus (HIV).
In the cytosol, there is a soluble proteolytic pathway that requires ATP and involves covalent conjugation of the cellular proteins with the small polypeptide ubiquitin (xe2x80x9cUbxe2x80x9d) (Hershko et al., A. Rev. Biochem. 61:761-807 (1992); Rechsteiner et al., A. Rev. Cell. Biol. 3:1-30 (1987)). Thereafter, the conjugated proteins are hydrolyzed by a 26S proteolytic complex containing a 20S degradative particle called the proteasome (Goldberg, Eur. J. Biochem. 203:9-23 (1992); Goldberg et al., Nature 357:375-379 (1992)). This multicomponent system is known to catalyze the selective degradation of highly abnormal proteins and short-lived regulatory proteins. However, the system also appears to be responsible for the breakdown of most proteins in maturing reticulocytes (Boches et al., Science 215:978-980 (1982); Spenser et al., J. Biol. Chem. 257:14122-14127 (1985)), in growing fibroblasts (Ciechanover et al., Cell 37:57-66 (1984); Gronostajski et al., J. Biol. Chem. 260:3344-3349 (1985)), and in atrophying skeletal muscle.
The first step in degradation of many proteins involves their conjugation to Ub by an ATP-requiring process. The ubiquitinated proteins are then degraded by an ATP-dependent proteolytic complex, referred to above, known as the 26S proteasome complex.
The precise nature of the 26S proteasome complex is unclear, although it has been shown that the 1000-1500 kDa (26S) complex can be formed in extracts of energy-depleted reticulocytes by an ATP-dependent association of three components, referred to as CF-1, CF-2, and CF-3 (Ganoth, D. et al., J. Biol. Chem. 263:12412-12419 (1988)). A large (xcx9c700 kDa) multimeric protease found in the cytoplasm and nucleus of eukaryotic cells, referred to as the proteasome, is a component (CF-3) (Driscoll et al., J. Biol. Chem. 265:4789-4792 (1992); Eytan et al., Proc. Natl. Acad. Sci. USA 86:7751-7755 (1989); Orlowski, Biochemistry 29:10289-10297 (1990) and Rivett, Arch. Biochem. Biophys. 268:1-8 (1989)).
The proteasome is believed to make up the catalytic core of the large 26S multisubunit cytoplasmic particle necessary for the ubiquitin-dependent pathway of intracellular proteolysis (Driscoll et al., J. Biol. Chem. 265:4789-4692 (1990); Eytan et al., Proc. Natl. Acad. Sci. U.S.A. 86:7751-7755 (1989); Hough et al., Biochemistry 262:8303-8313 (1987); McGuire et al., Biochim. Biophys. Acta 967:195-203 (1988); Rechsteiner et al., A. Rev. Cell. Biol. 3:1-30 (1987); Waxman et al., J. Biol. Chem. 262:2451-2457 (1987)). By itself, the proteasome is unable to degrade ubiquitinated proteins, but provides most of the proteolytic activity of the 26S proteasome complex.
There is another ATP-dependent protease that is involved in degradation of ubiquitinated proteins, forms a complex with the proteasome, and appears to be part of the 26S proteasome complex, which rapidly degrades proteins conjugated to ubiquitin. This protease, referred to as multipain, has been identified in muscle and plays an essential role in the ATP-ubiquitin-dependent pathway.
The complex formed between multipain and proteasome in vitro appears very similar or identical to the 1500 kDa Ub-conjugate, degrading enzyme, or 26S proteolytic complex, isolated from reticulocytes and muscle. The complexes contain the characteristic 20-30 kDa proteasome subunits, plus a number of larger subunits, including the six large polypeptides found in multipain. The complex formed contains at least 10-12 polypeptides of 40-150 kDa.
A 40 kDa polypeptide regulator of the proteasome, which inhibits the proteasome""s proteolytic activities has been purified from reticulocytes and shown to be an ATP-binding protein whose release appears to activate proteolysis. The isolated regulator exists as a 250 kDa multimer and is quite labile (at 42xc2x0 C.). It can be stabilized by the addition of ATP or a nonhydrolyzable ATP analog, although the purified regulator does not require ATP to inhibit proteasome function and lacks ATPase activity. The regulator has been shown to correspond to an essential component of the 1500 kDa proteolytic complex. The regulator appears identical to CF-2 by many criteria. These findings suggest that the regulator plays a role in the ATP-dependent mechanism of the 26S proteasome complex.
There is also a system in the cytosol that generates antigenic particles from endogenously synthesized cellular and viral proteins (Moore et al., Cell 54:777-785 (1988); Morrison et al., J. Exp. Med. 163:903-921 (1986); Powis et al., Nature 354:529-531 (1991); Spies et al., Nature 351:323-324 (1991); Townsend et al., Cell 42:457-467 (1985); Townsend et al., Nature 324:575-577 (1986); Monaco et al., Proc. Natl. Acad. Sci. U.S.A. 79:3001-3005 (1982); Monaco, Immun. Today 13:173-179 (1992); Yewdell et al., Adv. Immun. 52:1-123 (1992); Townsend et al., J. Exp. Med. 168:1211-1224 (1988)). Indirect evidence suggests a role for proteolytic particles closely resembling and perhaps identical to the proteasome (Goldberg et al., Nature 357:375-379 (1992); Monaco, Immun. Today 13:173-179 (1992); Parham, Nature 348:674-675 (1990); Yang et al., Proc. Natl. Acad. Sci. U.S.A. 89:4928-4932 (1992); (Brown et al., Nature 353:355-357 (1991)). It has been shown that the proteasome is responsible for cytoplasmic processing of MHC class I antigen molecules.
The 20S proteasome is composed of about 15 distinct 20-30 kDa subunits. It contains at least three different peptidases that cleave specifically on the carboxyl side of the hydrophobic, basic, and acidic amino acids (Goldberg et al., Nature 357:375-379 (1992); Goldberg, Eur. J. Biochem. 203:9-23 (1992); Orlowski, Biochemistry 29:10289-10297 (1990); Rivett et al., Archs. Biochem. Biophys. 218:1 (1989); Rivett et al., J. Biol. Chem. 264:12,215-12,219 (1989); Tanaka et al., New Biol. 4:1-11 (1992)). These peptidases are referred to as the chymotrypsin-like peptidase, the trypsin-like peptidase, and the peptidylglutamyl peptidase. Which subunits are responsible for these activities is unknown, although the cDNA""s encoding several subunits have been cloned (Tanaka et al., New Biol. 4:1-11 (1992)).
Recent studies have found that the 20S proteasomes resemble in size and subunit composition the MHC-linked LMP particles (Driscoll et al., Cell 68:823 (1992); Goldberg et al., Nature 357:375-379 (1992); Matthews et al., Proc. Natl. Acad. Sci. U.S.A. 86:2586 (1989); Monaco et al., Human Immunology 15:416 (1986); Parham, Nature 348:674-675 (1990); Martinez et al., Nature 353:664 (1991); Oritz-Navarette et al., Nature 353:662 (1991); Glynne et al., Nature 353:357 (1991); Kelly et al., Nature 353:667 (1991); Monaco et al., Proc. Natl. Acad. Sci. U.S.A. 79:3001 (1982); Brown et al., Nature 353:355 (1991); Goldberg, Eur. J. Biochem. 203:9-23 (1992); Tanaka et al., New Biol. 4:1-11 (1992)).
Various inhibitors of the peptidases of the proteasome have been reported (Dick et al., Biochemistry 30:2725-2734 (1991); Goldberg et al., Nature 357:375-379 (1992); Goldberg, Eur. J. Biochem. 203:9-23 (1992); Orlowski, Biochemistry 29:10289-10297 (1990); Rivett et al., Archs. Biochem. Biophys. 218:1 (1989); Rivett et al., J. Biol. Chem. 264:12,215-12,219 (1989); Tanaka et al., New Biol. 4:1-11 (1992)). These include known inhibitors of chymotrypsin-like and trypsin-like proteases, as well as inhibitors of thiol (or cysteine) and serine proteases. In addition, some endogenous inhibitors of proteasome activities have been isolated. These include the 240 kDa and the 200 kDa inhibitors isolated from human erythrocytes (Murakami et al., Proc. Natl. Acad. Sci. U.S.A. 83:7588-7592 (1986); Li et al., Biochemistry 30:9709-9715 (1991)) and purified CF-2 (Goldberg, Eur. J. Biochem. 203:9-23 (1992)). In addition to antibiotic inhibitors originally isolated from actinomycetes (Aoyagi et al., Proteases and Biological Control, Cold Spring Harbor Laboratory Press, pp. 429-454 (1975)), a variety of peptide aldehydes have been synthesized, such as the inhibitors of chymotrypsin-like proteases described by Siman et al. (WO 91/13904).
Novel molecules can also be obtained and tested for inhibitory activity. As illustrated by the above cited references, various strategies are known in the art for obtaining the inhibitors for a given protease. Compound or extract libraries can be screened for inhibitors using peptidase assays. Alternatively, peptide and peptidomimetic molecules can be designed based on knowledge of the substrates of the protease. For example, substrate analogs can be synthesized containing a reactive group likely to interact with the catalytic site of the protease (see, e.g., Siman et al., WO 91/13904; Powers et al., in Proteinase Inhibitors, Barrett et al. (eds.), Elsevier, pp. 55-152 (1986)). The inhibitors can be stable analogs of catalytic transition states (transition state analog inhibitors), such as Z-Gly-Gly-Leu-H, which inhibits the chymotrypsin-like activity of the proteasome (Orlowski, Biochemistry 29:10289-10297 (1990); see also Kennedy and Schultz, Biochemistry 18:349 (1979)).
Various natural and chemical protease inhibitors reported in the literature, or molecules similar to them, include peptides containing an xcex1-diketone or an xcex1-keto ester, peptide chloromethyl ketones, isocoumarins, peptide sulfonyl fluorides, peptidyl boronates, peptide epoxides, and peptidyl diazomethanes (Angelastro et al., J. Med Chem. 33:11-13 (1990); Bey et al., EPO 363,284; Bey et al., EPO 364,344; Grubb et al., WO 88/10266; Higuchi et al., EPO 393,457; Ewoldt et al., Molecular Immunology 29(6):713-721 (1992); Hernandez et al., Journal of Medicinal Chemistry 35(6): 1121-1129 (1992); Vlasak et al., Journal of Virology 63(5):2056-2062 (1989); Hudig et al., Journal of Immunology 147(4): 1360-1368 (1991); Odakc et al., Biochemistry 30(8):2217-2227 (1991); Vijayalakshmi et al., Biochemistry 30(8):2175-2183 (1991); Kam et al., Thrombosis and Haemostasis 64(1):133-137 (1990); Powers et al., Journal of Cellular Biochemistry 39(1):33-46 (1989); Powers et al., Proteinase Inhibitors, Barrett et al., Eds., Elsevier, pp. 55-152 (1986); Powers et al., Biochemistry 29(12):3108-3118 (1990); Oweida et al., Thrombosis Research 58(2):391-397 (1990); Hudig et al., Molecular Immunology 26(8):793-798 (1989); Orlowski et al., Archives of Biochemistry and Biophysics 269(1): 125-136 (1989); Zunino et al., Biochimica et Biophysica Acta. 967(3):331-340 (1988); Kam et al., Biochemistry 27(7):2547-2557 (1988); Parkes et al., Biochem J. 230:509-516 (1985); Green et al., J. Biol. Chem. 256:1923-1928 (1981); Angliker et al., Biochem. J. 241:871-875 (1987); Puri et al., Arch. Biochem. Biophys. 27:346-358 (1989); Hanada et al., Proteinase Inhibitors: Medical and Biological Aspects, Katunuma et al., Eds., Springer-Verlag pp. 25-36 (1983); Kajiwara et al., Biochem. Int. 15:935-944 (1987); Rao et al., Thromb. Res. 47:635-637 (1987); Tsujinaka et al., Biochem. Biophys. Res. Commun. 153:1201-1208 (1988)).
Various inhibitors of ubiquitin conjugation to proteins are also known (Wilkinson et al., Biochemistry 29:7373-7380 (1990)).
Certain peptide aldehydes and peptide xcex1-keto esters containing a hydrophobic residue in the P1 position were tested by Vinitsky et al. (Biochemistry 31:9421-9428 (1992), see also, Orlowski et al. Biochemistry 32:1563-1572 (1993)) as potential inhibitors of the chymotrypsin-like activity of the proteasome. Three peptide aldehydes, (benzyloxycarbonyl)-Leu-Leu-phenylalaninal (Z-LLF-H), N-acetyl-Leu-Leu-Norleucinal (Ac-LLnL-H), and N-acetyl-Leu-Leu-methioninal (Ac-LLM-H) were found to be slow binding inhibitors with Ki values of 0.46, 5.7, and 33 xcexcM, respectively. Of the several peptide xcex1-keto ester inhibitors tested, Z-Leu-Leu-Phe-COOEt was the most potent inhibitor of the chymotrypsin-like activity with a Ki of 53 xcexcM. Many such compounds exist.
Other tripeptides that have been described in the literature include Ac-Leu-Leu-Leu-H, Ac-Leu-Leu-Met-OR, Ac-Leu-Leu-Nle-OR, Ac-Leu-Leu-Leu-OR, Ac-Leu-Leu-Arg-H, Z-Leu-Leu-Leu-H, Z-Arg-Leu-Phe-H, and Z-Arg-Ile-Phe-H, where OR, along with the carbonyl of the preceding amino acid residue, represents an ester group.
Goldberg, in U.S. patent application Ser. No. 07/699,184, filed May 13, 1991, now U.S. Pat. No. 5,340,736 discloses that the ATP-ubiquitin-dependent process has been shown to be responsible for the excessive protein degradation that occurs in conditions or disease states in which there is severe loss of body mass and negative nitrogen balance. A method of inhibiting the accelerated or enhanced proteolysis, a method of identifying inhibitors of the process, multipain and proteasome inhibitors are also disclosed.
Goldberg et al., in U.S. patent application Ser. No. 08/016,066, filed Feb. 10, 1993, now abandoned disclose methods and drugs that inhibit the processing of antigens for presentation by major histocompatibility complex class I molecules. Specifically, inhibitors of the ATP-ubiquitin-dependent proteolytic pathway are described, which can inhibit MHC-I antigen presentation. These methods and drugs may be useful for the treatment of autoimmune diseases and for reducing rejection of organs and graft transplants. See, also, Michalek et al., Nature 363:552-554 (1993).
Tsubuki et al., Biochem. and Biophys. Res. Commun. 196(3):1195-1201 (1993) reported that a tripeptide aldehyde protease inhibitor, benzyloxycarbonyl(Z)-Leu-Leu-leucinal, initiates neurite outgrowth in PC12 cells at an optimal concentration of 30 nM. The following synthetic peptides are also mentioned: Z-Leu-Leu-Gly-H, Z-Leu-Leu-Ala-H, Z-Leu-Leu-Ile-H, Z-Leu-Leu-Val-H, Z-Leu-Leu-Nva-H, Z-Leu-Leu-Phe-H, Z-Leu-Leu-Leu-H, Bz-Leu-Leu-Leu-H, Ac-Leu-Leu-Leu-H, Z-Leu-Leu-Leu.sc, Z-Leu-Leu-Leu.ol, Z-Leu-Leu-Leu, Dns-Leu-Leu-Leu-H, Dns-Leu-Leu-Leu-CH2Cl, and Leupeptin.
Siman et al. (WO 91/13904) disclose chymotrypsin-like proteases and their inhibitors. The inhibitors have the formula R-A4-A3-A2-Y, wherein
R is hydrogen, or a N-terminal blocking group;
A4 is a covalent bond, an amino acid or a peptide;
A3 is a covalent bond, a D-amino acid, Phe, Tyr, Val, or a conservative amino acid substituent of Val;
A2 is a hydrophobic amino acid or lysine or a conservative amino acid substituent thereof, or when A4 includes at least two amino acids, A2 is any amino acid; and
Y is a group reactive with the active site of said protease.
Powers (WO 92/12140) discloses peptide ketoamides, ketoacids, and ketoesters and their use in inhibiting serine proteases and cysteine proteases.
Bartus et al. (WO 92/1850) disclose uses for Calpain inhibitor compounds and pharmaceutical compositions containing them. One use of these compounds is in the treatment of a neurodegenerative pathology in a human patient. The disclosure also provides additional uses and pharmaceutical compositions containing Calpain inhibitor compounds, such as peptide ketoamides, peptide ketoacids, and peptide ketoesters.
The present invention relates to a method for reducing the cellular content and activity of NF-xcexaB.
In a preferred embodiment, the present invention relates to a method for reducing the cellular content and activity of NF-xcexaB in an animal comprising contacting cells of the animal with inhibitors of proteasome function or ubiquitin conjugation.
More particularly, the present invention is directed to a method for reducing the cellular content and activity of NF-xcexaB in an animal comprising contacting cells of the animal with a proteasome function or ubiquitin conjugation inhibitor of the structure (1): 
where
P is an amino-group-protecting moiety;
B1, B2, B3, and B4 are independently selected from the group consisting of 
xe2x80x83X1, X2, and X3 are independently selected from the group consisting of 
xe2x80x83and xe2x80x94CHxe2x95x90CHxe2x80x94;
R is a hydrogen, alkyl, acyl, or carboxyl;
R1, R2, R3, and R4 are independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, and xe2x80x94CH2xe2x80x94R5,
where R5 is aryl, aralkyl, alkaryl, cycloalkyl or xe2x80x94Yxe2x80x94R6,
where Y is a chalcogen, and R6 is alkyl; and
A is 0 or 1.
The xe2x80x9canimalsxe2x80x9d referred to herein are preferably mammals. Both terms are intended to include humans.